Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions

Abstract

Lactate has long been considered a "waste" by-product of cell metabolism, and it accumulates at sites of inflammation. Recent findings have identified lactate as an active metabolite in cell signalling, although its effects on immune cells during inflammation are largely unexplored. Here we ask whether lactate is responsible for T cells remaining entrapped in inflammatory sites, where they perpetuate the chronic inflammatory process. We show that lactate accumulates in the synovia of rheumatoid arthritis patients. Extracellular sodium lactate and lactic acid inhibit the motility of CD4+ and CD8+ T cells, respectively. This selective control of T cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1) that we find selectively expressed by CD4+ and CD8+ subsets, respectively. We further show both in vitro and in vivo that the sodium lactate-mediated inhibition of CD4+ T cell motility is due to an interference with glycolysis activated upon engagement of the chemokine receptor CXCR3 with the chemokine CXCL10. In contrast, we find the lactic acid effect on CD8+ T cell motility to be independent of glycolysis control. In CD4+ T helper cells, sodium lactate also induces a switch towards the Th17 subset that produces large amounts of the proinflammatory cytokine IL-17, whereas in CD8+ T cells, lactic acid causes the loss of their cytolytic function. We further show that the expression of lactate transporters correlates with the clinical T cell score in the synovia of rheumatoid arthritis patients. Finally, pharmacological or antibody-mediated blockade of subtype-specific lactate transporters on T cells results in their release from the inflammatory site in an in vivo model of peritonitis. By establishing a novel role of lactate in control of proinflammatory T cell motility and effector functions, our findings provide a potential molecular mechanism for T cell entrapment and functional changes in inflammatory sites that drive chronic inflammation and offer targeted therapeutic interventions for the treatment of chronic inflammatory disorders.

Fig 1. Lactate inhibits T cell motility.

(A) Lactate measurements in the synovial fluid of osteoarthritis (OA) or RA patients. (B–C) In vitro chemotaxis of activated CD4⁺ (B) and CD8⁺ (C) T cells towards CXCL10 (300 ng/ml) in the presence of lactic acid (10mM) or sodium lactate (10 mM) shown as kinetic (left panel) and 4 h time point (right panel). (D) In vitro chemotaxis of activated CD4⁺ T cells towards CXCL10 (300 ng/ml) in the presence of increasing concentration of sodium lactate shown as kinetic (left panel) and 4 h time point (right panel). (E) In vitro chemotaxis (4 h time point) of activated CD8⁺ T cells towards CXCL10 (300 ng/ml) in the presence of sodium lactate (10 mM) or HCl (pH 4.5) alone, or sodium lactate in combination with increasing concentrations of HCl to obtain progressively reduced pH as indicated in figure. (A) OA, n = 4 and RA n = 8. (B right panel) n = 4. (C–D right panel, E) n = 3. (B–D left panel) Data is representative of three independent experiments; the underlying numerical data and statistical analysis for each independent experiment can be found in the supporting file, S1 Data, Fig 1B–1D. (A–E) Underlying numerical data and statistical analysis can be found in the supporting file, S1 Data, Fig 1A–1E. Values denote mean ± standard deviation (SD). * p <0.05; ** p <0.01; *** p <0.001.

Fig 2. Sodium lactate and lactic acid act on CD4⁺ and CD8⁺ T cell subsets, respectively, through specific cell membrane transporters.

(A) Total protein levels of the transporters Slc16a1 and Slc5a12 as assessed by western blot in activated CD4⁺ and CD8⁺ T cell subsets. (B–D) In vitro chemotaxis (4 h time point) of activated CD8⁺ T cells towards CXCL10 (300 ng/ml) in the presence of lactic acid (10 mM) alone, or in combination with α-cyano-4-hydroxycinnamate (CHC) (425 μM), phloretin (25 μM), or anti-Slc16a1 antibody (2.5 μg/ml) (B), or increasing concentrations of AR-C155858 as indicated in the figure (C), and activated CD4⁺ T cells towards CXCL10 (300 ng/ml) in the presence of sodium lactate (10 mM) alone, or in combination with an anti-Slc5a12 antibody (2.5 μg/ml) or two specific short hairpin RNAs (shRNAs) (D). An isotype control antibody has been included to control for antibody specificity (B, D), and a nonspecific shRNA has been included to control for gene knockdown specificity (D). (B–D) n = 3. Underlying numerical data and statistical analysis can be found in the supporting file, S1 Data, Fig 2B–2D. Values denote mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3. Basal and chemokine-induced aerobic glycolysis is required for CD4⁺ T cell migration.

(A) Western blots with antibodies against Hk1, aldolase A, PkM1/2, enolase 1, and β-actin in activated CD4⁺ T cells treated with CXCL10 (1,000 ng/ml) alone or in combination with sodium lactate (10 mM), or left untreated. Densitometric quantification of western blots denotes mean ± SD, n = 3 (with biological replicates run in duplicate). *p < 0.05; ***p < 0.001. (B) Relative mRNA expression levels of Hk1, PkM2, and glucose transporters (Glut1, Glut2, Glut3, Glut4) in activated CD4⁺ T cells 6 h post-treatment with CXCL10 (1,000 ng/ml) as assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). mRNA levels in naive CD4⁺ T cells were set to 1. (C) Extracellular acidification rate (ECAR) trace of glycolytic activity expressed as mpH/min in activated CD4⁺ T cells treated with sodium lactate (10 mM) or phosphate buffered saline (PBS). Vertical lines represent addition times of sodium lactate or PBS, respectively. (D) Measurements of glucose uptake and flux in activated CD4⁺ T cells pretreated with 2-deoxyglucose (2-DG) (1 mM), sodium-lactate (10 mM) or lactic acid (10 mM) and then incubated with the fluorescent probes 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (6-NBDG) or 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG). (E) ECAR trace of glycolytic activity in activated CD4⁺ T cells treated with CXCL10 (1,000 ng/ml) and sodium lactate (10 mM). Vertical lines represent the addition times of CXCL10, sodium lactate and PBS. (F) In vitro chemotaxis (4 h time point) towards CXCL10 (300 ng/ml) of activated CD4⁺ T cells pretreated with Rapamycin (200 nM), 2-DG (1 mM) or Metformin (2 mM). (G) Relative enrichment of IV-injected activated CD4⁺ T cells pretreated with Rapamycin (200 nM), 2-DG (1 mM) or Metformin (2 mM) and subsequently labelled with 7-Hydroxy-9H-(1,3-Dichloro-9,9-Dimethylacridin-2-One (DDAO) cell fluorescent dye in the peritoneal lavage of syngeneic recipient C57BL/6 mice i.p. injected with CXCL10 (120 ng/mouse). (H) Spontaneous transendothelial migration (6 h time point) of activated CD4⁺ T cells in the presence of rapamycin (200nM), 2-DG (1 mM) or metformin (2 mM). (B–C, E) Data is representative of three independent experiments; the underlying numerical data and statistical analysis for each independent experiment can be found in the supporting file, S1 Data, Fig 3B–3C, 3E. (D, F, H) n = 3. (G) n = 4. (A–H) Underlying numerical data and statistical analysis can be found in the supporting file, S1 Data, Fig 3A–3H. Values denote mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4. Lactate modulates effector T cell functions.

(A) Relative mRNA expression levels of the cytokines interferon-gamma (Ifn-γ), Tnf-β, Il-4, Il-5, Il-13, and Il-17 and of the transcription factor Rorc as assessed by qRT-PCR in CD4⁺ subsets Th0, Th1, Th2, and Th17 treated with sodium lactate (10 mM) or left untreated. mRNA levels of each cytokine expressed by untreated Th0 cells were set to 1. (B) Intracellular staining of IL-17A and IFN-γ in activated CD4⁺ T cells treated with sodium lactate (10 mM) or left untreated. (C) Relative mRNA expression levels of Il-17 and Rorc in activated CD4⁺ T cells treated with sodium lactate alone or in combination with an anti-Slc5a12 antibody. mRNA levels of untreated T cells were set to 1. (D) Cell survival of allogeneic endothelial cells in the presence of CD8⁺ cytotoxic T cells and lactic acid (10 mM) or sodium lactate (10 mM) shown as kinetic (left panel) and 6 h time point (right panel). (A, C, D left panel) Data is representative of three independent experiments; the underlying numerical data and statistical analysis for each independent experiment can be found in the supporting file, S1 Data, Fig 4A, 4C, and 4D. (B, D right panel) n = 3. (A–D) Underlying numerical data and statistical analysis can be found in the supporting file, S1 Data, Fig 4A–4D. Values denote mean ± SD. *p < 0.05; **p < 0.01.

Fig 5. High Slc5a12 expression in RA in humans.

(A) Representative images of RA synovial tissues stained for CD3 displaying progressively higher degree of T cell infiltration as quantified using a semiquantitative score from T0 (absence of infiltrating T-cells) to T3 (large number of infiltrating T cells organizing in ectopic follicles) as shown in [35]. (B) Relative mRNA expression levels of Slc16a1 and Slc5a12 in the synovial fluid isolated from the joints of RA patients. Samples are grouped based on their T cell score as described in A. Values denote mean ± SD, (T0) n = 6 and (T2–3) n = 7. *p < 0.05. (C) Double immunofluorescence staining for Slc5a12 and CD4 or CD8 in the synovial tissue of RA patients. Slc5a12 (green) is highly expressed within the RA synovia in the presence of a high degree of CD4⁺ (red) T cell infiltration. Merging (yellow) of the green and red channels demonstrates that Slc5a12 is selectively expressed by CD4⁺ but not CD8⁺ infiltrating T cells. Quantification of the % double positive cells is provided upon counting positive cells (single and double positive for each marker) in at least 6 images per condition. Columns represent % of double positive CD4⁺ Slc5a12⁺ population within the CD4⁺ or Slc5a12⁺ cells and % of double positive CD8⁺ Slc5a12⁺ population within the CD8⁺ or Slc5a12⁺ cells. Scale bars: 50 μm. (D) In vitro chemotaxis (4 h time point) of activated human CD4⁺ and CD8⁺ T cells towards CXCL10 (300 ng/ml) in the presence of lactic acid (10 mM) or sodium lactate (10 mM). (E) Intracellular staining of IL-17A in activated human CD4⁺ T cells treated with sodium lactate (10 mM) or left untreated. (D) n = 3. (E) n = 4. (B–E) Underlying numerical data and statistical analysis can be found in the supporting file, S1 Data, Fig 5B–5E. Values denote mean ± SD. *p < 0.05.

Fig 6. Inhibition of lactate transporters promotes the release of T-cells from the inflamed site in zymosan-induced peritonitis.

(A) Lactate levels in the peritoneum of zymosan-treated mice. (B) Number of CD4⁺ and CD8⁺ T cells, respectively, in the peritoneal lavage of C57BL/6 mice injected i.p. with zymosan (1 mg/mouse) to induce peritonitis, and 5 d later, i.p. treated with phloretin (50 μM), an anti-Slc5a12 antibody (5 μg/ml) or an isotype control antibody. (C) Number of carboxyfluorescein succinimidyl ester (CFSE)-labeled activated CD4⁺ T cells in the peritoneal lavage (left panel) or spleen (right panel), respectively, of C57BL/6 mice injected i.p. with zymosan (1 mg/mouse), then i.p. treated with phloretin (50 μM), an anti-Slc5a12 specific antibody (5μg/ml) or an isotype control antibody. (A–C) n = 3 or more. Underlying numerical data and statistical analysis can be found in the supporting file, S1 Data, Fig 6A–6C. Values denote mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 7. Schematic of the proposed mechanism of lactate effects on T cells in the inflammatory site.

(A) The motility of CD4⁺ and CD8⁺ T cells is blocked once they get exposed to elevated levels of lactate in the inflammatory site. Lactic acid also causes loss of cytolytic activity by CD8⁺ T cells, and sodium lactate promotes the production of IL-17 by CD4⁺ T cells. (B) Pharmacologic targeting of lactate transporters re-establish T cell migration away from the inflammatory site and block the production of high amounts of IL-17.